N- and O-glycans are attractive clinical biomarkers as glycosylation changes in response to diseases. The limited availability of defined clinical specimens impedes glyco-biomarker identification and validation in large patient cohorts. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are the common form of sample preservation in clinical pathology, but qualitative and quantitative N- and O-glycomics of such samples has not been feasible to date. Here, we report a highly sensitive and glycan isomer selective method for simultaneous N- and O-glycomics from histopathological slides. As few as 2000 cells isolated from FFPE tissue sections by laser capture microdissection were sufficient for in-depth histopathology- glycomics using porous graphitized carbon nanoLC ESI-MS/MS. N- and O-glycan profiles were similar between unstained and hematoxylin and eosin stained FFPE samples but differed slightly compared with fresh tissue. This method provides the key to unlock glyco-biomarker information from FFPE histopathological tissues archived in pathology laboratories worldwide.